We will follow up on signals from a genome wide association study (GWAS) of oral clefts now being conducted with support from UOl-DE-004425;"International Consortium to Identify Genes &Interactions Controlling Oral Clefts", 2007-2009;TH Beaty. Oral clefts are among the most common human birth defects and have a complex and heterogeneous etiology. Genotyping for this project should be completed in early 2009 and our analysis will identify genes influencing risk directly those acting only in the presence of an environmental risk factor, and/or genes showing measurable parent-of-origin effects which may represent imprinting. In this response to the FaceBase initiative (RFA-DE-09-003) we will build upon our GWAS results by using high throughput sequencing (HTS) techniques of genes/regions yielding statistical evidence of linkage and disequilibrium (Aim 1) and to conduct functional genomic studies of these same genes to understand how they act in vivo using the zebrafish model (Aim 2). We will first focus on genes identified through analysis of single nucleofide polymorphic (SNP) markers from our GWAS and will use HTS to identify all variants (rare mutations and novel markers) that may cause oral clefts. We will then undertake a systematic analysis of 60.000 markers in regions of known copy number variants (CNV) available on this platform, to test and sequence genes that may influence risk through structural variation. In our Aim 2, we will use the zebrafish for expression profiling and loss-of-function analyses of genes identified from our GWAS and sequenced in Aim 1. We will perform further functional studies including expression array, miRNA and/or ChlP-seq analysis. This functional genomics work will identify genes expressed in zebrafish head mesenchyme, cranial neural crest cells, pharyngeal arches, neurocranium and/or other relevant tissues using in situ hybridization and cartilage/bone staining methods in normal embryos and knockdown morphants. We will also gather further functional information via global gene and miRNA expression profiles to identify possible networks or pathways involving interacting genes, and explore methylation/imprinting miRNA regulation and/or chromatin targeting of selected genes. PUBLIC HEALTH RELEVANCE: This project is designed to follow up evidence from a genome wide study of oral clefts (cleft lip, cleft palate &cleft lip and palate) in a large international study of humans by sequencing specific genes to identify causal variants/mutations and using an animal model to better define how these genes function in a living animal.